Rossella Monte

Immunoglobulin A response against Gardnerella vaginalis hemolysin and sialidase activity in bacterial vaginosis

OBJECTIVE The aim of this study was to investigate the correlation between the immunoglobulin A immune response to Gardnerella vaginalis hemolysin and sialidase activity in vaginal fluids from patients with bacterial vaginosis. STUDY DESIGN Nonpregnant women who were examined at a gynecologic clinic, in an age range of 18 to 62 years, were enrolled. The study population comprised 131 healthy volunteers, 32 women with bacterial vaginosis that was positive for immunoglobulin A to Gardnerella vaginalis hemolysin, 40 women with bacterial vaginosis that was negative for immunoglobulin A to Gardnerella vaginalis hemolysin, and 19 women with Candida vaginitis. Bacterial vaginosis was diagnosed by clinical criteria and Gram stain. RESULTS Sialidase activity was present in 75% (54/72) of patients with bacterial vaginosis. Women having bacterial vaginosis and lacking a specific immunoglobulin A response had a significantly higher level of sialidase activity than patients who had an immune response against Gardnerella vaginalis hemolysin. Sialidase activity was detected in 87% (35/40) of the former subgroup of patients with bacterial vaginosis and in 59% (19/32) of women of the latter subgroup. No sialidase activity was measured in patients with candidiasis. Specificity of the assay for healthy controls was 95% (124/131 women without sialidase activity). CONCLUSIONS Sialidases produced by Prevotella bivia and other microorganisms present in the microflora of patients with bacterial vaginosis are very likely a virulence factor not only by destroying the mucins and enhancing adherence of bacteria but also by impairing a specific immunoglobulin A immune response against other virulence factors such as cytotoxin from Gardnerella vaginalis.

Impairment of the Mucosal Immune System: IgA and IgM Cleavage Detected in Vaginal Washings of a Subgroup of Patients with Bacterial Vaginosis

The integrity of the immunoglobulins in vaginal washings of patients with bacterial vaginosis was examined to answer the question of the lack of immune response against Gardnerella vaginalis cytolysin. Clinically diagnosed patients (n=100) were recruited and their vaginal washings examined by Western blotting. Many showed IgA and IgM partially or extensively degraded. According to the degradation pattern, the patients were subdivided into 4 subsets, from intact (score 0) to completely degraded IgA (score +3). Statistical analysis of the data showed a correlation between IgA degradation and absence of immune response to G. vaginalis cytolysin. The extent of IgA degradation correlated also with the sialidase (but not with the prolidase) activity level. All women showed intact IgG and human serum albumin and no trypsin-like activity. Patients with bacterial vaginosis having high sialidase activity and extensive IgA degradation in their secretions could incur more dangerous infections and adverse pregnancy outcomes.

Pore‐forming and haemolytic properties of the Gardnerella vaginalis cytoiysin

The pleomorphic bacterium Gardnerella vaginalis releases in the culture broth a haemolytic exotoxin (Gvh) which is probably a virulence determinant of this unique bacterium, implicated in gynaecological and urological disorders. This 59kDa cytolysin was purified to homogeneity in just one chromatographic step directly from the culture supernatant, a final specific activity up to 1.9 × 106 HU mg−1 being obtained. The toxin‐induced lesion on human erythrocytes results from the formation of a pore whose radius is approximately 2.4 nm. The damage is inhibited by osmotic protectants and shows a sigmoidal dose‐response profile suggesting an aggregation process of haemolysin molecules on the target membrane to create the functional lesion. The extent and the kinetics of haemolysis are strongly dependent on temperature and an activation energy of 64.0 kJ mol−1 has been derived. Lipid membranes can be very efficient inhibitors of Gvh‐haemolysis, being able to bind the toxin quite avidly. The inhibitory effect requires the presence of cholesterol and it is stronger when cholesterol is mixed with negatively charged phospholipids rather than with zwitterionic phospholipids, suggesting that a negative surface potential increases the affinity of the toxin for the lipid bilayer. The functional properties of Gvh have been compared with those of Clostridium perfringens theta‐toxin (PFO) and Escherichia coli haemolysin (HlyA), which are representative of widespread haemolysins produced by Gram‐positive and Gram‐negative bacteria, respectively. The toxin shares several features with the family of the so‐called ‘sulphydryl‐activated’ cytolysins produced by Gram‐positive bacteria, although Gvh does not truly belong to this family, being deactivated by β‐mercaptoethanol and being antigenically distinct from them. We report here for the first time the detection in the vaginal fluid of infected women of a specific IgA response against the toxin.

Specific immune response against Gardnerella vaginalis hemolysin in patients with bacterial vaginosis

OBJECTIVE Our goal was to study the mucosal host response in bacterial vaginosis by evaluating the presence of a specific immune response elicited against the Gardnerella vaginalis hemolysin in vaginal fluids of patients and by verifying its correlation with usual criteria adopted to diagnose bacterial vaginosis. STUDY DESIGN A total of 123 white women attending the gynecologic care unit for urogenital complaints or for screening of uterine malignancies (Papanicolaou test) aged from 20 to 60 years, nonmenstruating, were enrolled. Bacterial vaginosis was diagnosed by clinical criteria and a Gram stain score > 6. RESULTS We performed the determination of the antibody response in vaginal fluid against the hemolysin produced by G. vaginalis, a common agent present in bacterial vaginosis. The purified G. vaginalis toxin was a suitable antigen for detecting the presence of an immune response in the vaginal fluids of patients with bacterial vaginosis regardless of the strain of G. vaginalis present. A specific immunoglobulin A response was detected in 60% of women with overt bacterial vaginosis (Gram stain score > 6) and in 18.5% of women with intermediate vaginal flora (Gram stain score 4 to 6). The specificity of the test was 91%. CONCLUSIONS We found a correlation between the specific local immune response to G. vaginalis toxin and bacterial vaginosis. The highly purified form of the toxin is able to discriminate disorders from the opportunistic colonization by G. vaginalis.

Nitrate transport in cucumber leaves is an inducible process involving an increase in plasma membrane H+-ATPase activity and abundance

BackgroundThe mechanisms by which nitrate is transported into the roots have been characterized both at physiological and molecular levels. It has been demonstrated that nitrate is taken up in an energy-dependent way by a four-component uptake machinery involving high- and low- affinity transport systems. In contrast very little is known about the physiology of nitrate transport towards different plant tissues and in particular at the leaf level.ResultsThe mechanism of nitrate uptake in leaves of cucumber (Cucumis sativus L. cv. Chinese long) plants was studied and compared with that of the root. Net nitrate uptake by roots of nitrate-depleted cucumber plants proved to be substrate-inducible and biphasic showing a saturable kinetics with a clear linear non saturable component at an anion concentration higher than 2 mM. Nitrate uptake by leaf discs of cucumber plants showed some similarities with that operating in the roots (e.g. electrogenic H+ dependence via involvement of proton pump, a certain degree of induction). However, it did not exhibit typical biphasic kinetics and was characterized by a higher Km with values out of the range usually recorded in roots of several different plant species. The quantity and activity of plasma membrane (PM) H+-ATPase of the vesicles isolated from leaf tissues of nitrate-treated plants for 12 h (peak of nitrate foliar uptake rate) increased with respect to that observed in the vesicles isolated from N-deprived control plants, thus suggesting an involvement of this enzyme in the leaf nitrate uptake process similar to that described in roots. Molecular analyses suggest the involvement of a specific isoform of PM H+-ATPase (CsHA1) and NRT2 transporter (CsNRT2) in root nitrate uptake. At the leaf level, nitrate treatment modulated the expression of CsHA2, highlighting a main putative role of this isogene in the process.ConclusionsObtained results provide for the first time evidence that a saturable and substrate-inducible nitrate uptake mechanism operates in cucumber leaves. Its activity appears to be related to that of PM H+-ATPase activity and in particular to the induction of CsHA2 isoform. However the question about the molecular entity responsible for the transport of nitrate into leaf cells therefore still remains unresolved.

Transcriptomic analysis highlights reciprocal interactions of urea and nitrate for nitrogen acquisition by maize roots

Even though urea and nitrate are the two major nitrogen (N) forms applied as fertilizers in agriculture and occur concomitantly in soils, the reciprocal influence of these two N sources on the mechanisms of their acquisition are poorly understood. Therefore, molecular and physiological aspects of urea and nitrate uptake were investigated in maize (Zea mays), a crop plant consuming high amounts of N. In roots, urea uptake was stimulated by the presence of urea in the external solution, indicating the presence of an inducible transport system. On the other hand, the presence of nitrate depressed the induction of urea uptake and, at the same time, the induction of nitrate uptake was depressed by the presence of urea. The expression of about 60,000 transcripts of maize in roots was monitored by microarray analyses and the transcriptional patterns of those genes involved in nitrogen acquisition were analyzed by real-time reverse transcription-PCR (RT-PCR). In comparison with the treatment without added N, the exposure of maize roots to urea modulated the expression of only very few genes, such as asparagine synthase. On the other hand, the concomitant presence of urea and nitrate enhanced the overexpression of genes involved in nitrate transport (NRT2) and assimilation (nitrate and nitrite reductase, glutamine synthetase 2), and a specific response of 41 transcripts was determined, including glutamine synthetase 1-5, glutamine oxoglutarate aminotransferase, shikimate kinase and arogenate dehydrogenase. Also based on the real-time RT-PCR analysis, the transcriptional modulation induced by both sources might determine an increase in N metabolism promoting a more efficient assimilation of the N that is taken up.

Involvement of plasma membrane H+-ATPase in nitrate uptake by maize genotypes

The relationship between NO3 − uptake and root plasma membrane H+ ATPase (pmH+-ATPase) was investigated in maize genotypes. Seedlings were grown at different NO3− concentrations with or without NH4 + over a 24–48 h period. NO3 − uptake rates and (pmH+-ATPase) activities and amounts were monitored. Results show a close correlation between the changes of net NO3 − uptake and those of (pmH+-ATPase) activity. This behaviour was also found when inbred lines with different nitrate use efficiency (NUE) were tested.

Respuesta inmune específica contra hemolisina de Gardnerella vaginalis en pacientes con vaginosis bacteriana

Objetivo: Estudiar la respuesta mucosa del hospedero en vaginosis bacteriana evaluando la presencia de una respuesta inmune especifica contra la hemolisina de Gardnerella vaginalis en fluidos vaginales de pacientes y verificar su correlacion con los criterios adoptados para diagnosticar la vaginosis bacteriana. Diseno del estudio: Se incluyeron un total de 123 mujeres blancas que consultaron a una unidad de cuidado ginecologico por quejas urogenitales o para tamizaje de cancer uterino (prueba de Papanicolaou), con edades entre 20 y 60 anos y no menstruando. La vaginosis bacteriana se diagnostico por criterios clinicos y un puntaje de la tincion de Gram > 6. Resultados: Se determino la respuesta de anticuerpos en el fluido vaginal contra la hemolisisna producida por G. vaginalis, un agente comun presente en la vaginosis bacteriana. La toxina purificada de G. vaginalis fue un antigeno util para detectar la presencia de una respuesta inmune en los fluidos vaginales de las pacientes con vaginosis bacteriana independientemente de la cepa de G. Vaginalis presente. Se detecto una respuesta especifica de inmunoglobulina A en el 60% de las mujeres con vaginosis bacteriana evidente (puntaje de la tincion de Gram > 6) y en 18.5% de las mujeres con flora vaginal intermedia (puntaje de la tincion de Gram 4 a 6). La especificidad de la prueba fue de 91%. Conclusiones: Encontramos una correlacion entre la respuesta inmune local especifica contra la toxina de G. vagainalis y la vagnosis bacteriana. La forma altamente purificada de la toxina permite discriminar los desordenes de la colonizacion oportunista por G. vaginalis.